ABSTRACT: Tobacco plants (Nicotiana tabacum 'Xanthi') were transformed with a binary vector containing the coat protein gene of tomato mottle begomovirus (ToMoV) (Geminiviridae) modified by the deletion of 30 nucleotides in the 5' end. The R1 generation was screened for resistance against ToMoV, by inoculation with viruliferous whiteflies. Symptom development was recorded every week for 16 weeks using a five level visual scale, and ToMoV infection was confirmed by PCR and ELISA. The response to ToMoV infection was variable, wild type, enhanced wild type, immunity and recovery phenotypes were observed. Some transformed plants developed an unusual bright vein yellowing. All plants transformed with a binary vector lacking the truncated ToMoV coat protein gene were susceptible to ToMoV infection. Southern blot analysis of immune R1 plants showed the presence of multiple copies of the transgene in 80% of the plants tested. The transgene transcript was detected by northern blot analysis, but the transgene product was not detected by western blot analysis using antisera reactive with ToMoV coat protein. Selected resistant R1 transgenic plants were also screened for resistance against tobacco mosaic tobamovirus, cucumber mosaic cucumovirus, and tomato yellow leaf curl begomovirus. Disease development was evaluated by visual assessment of symptoms and the presence of the viruses was determined by ELISA or dot blot hybridization. The transgenic plants evaluated did not show resistance to any of the three viruses indicating a narrow breadth of resistance.

Summary:

ABSTRACT (cont.): The R2 generation of selected R1 ToMoV-resistant and susceptible plants was monitored and assessed for resistance to ToMoV. Disease development was monitored for 60 days using a five level visual scale and ToMoV infection was confirmed by PCR. Some R2 plants displayed attenuation of symptoms but recovery and immunity phenotypes were not observed. The transgene transcript was not be detected by northern blot hybridization in resistant or susceptible R2 plants. The sequence of the transgene from the R1 and R2 generations of a resistant plant were determined, and no mutations were found. The resistance appears to be a form of RNA-mediated resistance.

Figure 2-3. Hybridization analysis of total RNA from selected resistant transgenic
plants. The plants used in this assay did not show disease symptoms and tested negative
for the presence of ToMoV by PCR. The hybridization was performed with a biotin-
labeled probe to the full length ToMoV coat protein gene. A) Lane Icorresponds to a virus
infected non-transgenic plant, lane 3 corresponds to a non-inoculated, non-transformed
plant, lanes 2 and 4 correspond to resistant plants of lines Rl-cp 8 and Rl-cp 11. B) Lanel
corresponds to a non-inoculated, non-transformed tobacco plant ; lanes 2, 3, 4, and 5,
correspond to selected resistant plants of lines R1-cp 6, R1-cp 10, R1-cp 11, and R1-cp 12
showing the presence of the transgene transcript.

Table 3-1. Presence of the transgene in the R2 generation of selected resistant and
susceptible R, tobacco plants transformed with a truncated coat protein of ToMoV

Germplasm
line

Plant ID
Number

Number of R2
Plants tested

Number of R2
Plants with
Transgeneb

R, -cp 2

R, -cp 4

R, -cp 5

R, -cp 6

9 12

R, -cp 7

Table 3-1. Continuation

Germplasm
line

R1 -cp 8

R1 -cp 9

R1-cp 10

RI -cp 11

Plant ID
number

12

4

8
10

9

11

Number of R2
plants
inoculated

12

12

12
12

12

Number of R2
plants with the
transgeneb

6

6

6
9

6

a A consecutive number from 1 to 12 was given to each one of the twelve R, plants from
each line that were inoculated with ToMoV.

b The presence of the transgene was established by PCR amplification using primers JAP
28 and JAP 82 which anneal to the CaMV 35S promoter and the coat protein gene
coding region.

Table 3-2. Phenotypes observed in the R2 generation of tobacco plants transformed
with a truncated ToMoV coat protein gene at 60 days after inoculation with ToMoV

Germplasm
line

Number of
plants
evaluated

Number of
plants with
attenuated
symptoms a

Number of plants
with severe
symptoms b

Number of
asymptomatic
plants

R2-cp 6 01 12 3 8 1

R2-cp 6 04 12 3 7 2

R2-cp 6 06 12 2 8 2

R2-cp 6 08 12 6 6 0

R2-cp 7 09 12 0 12 0

R2-cp 10- 10 12 1 10 1

R2-cp 11- 06 12 0 12 0

non-
transgenic 12 0 11 1
controls

a Symptom expression in transgenic and non-transgenic plants were rated using a 5 level
visual scale. At 60 dai control plants displayed symptoms rated 3. Plants with a
symptom severity rating of 1 or 2 were considered to display attenuated symptoms.